The formation of cancer is a multi-stage process that involves a series of molecular and structural alterations. Solid tumours, such as melanoma, lung, colon, head/neck, ovary, breast, pancreatic and prostate cancers, account for 90% of all cancers. The hallmarks of solid tumours at a late stage are extensive angiogenesis, necrosis and hypoxia along with the distinct presence of a central (inner) region and a peripheral region. The central region of a solid tumour characteristically has high interstitial pressure, low extracellular pH, tumour cell heterogeneity and anaerobic metabolisms, including glycolysis with high glucose concentration and lactate state. The peripheral region typically contains rapidly dividing cancer cells although these cells only account for 25-50% of the total cells present in the peripheral region. The remaining cells in the peripheral region are typically non-cancerous stroma cells.
The tumour inner central environments are detrimental for current chemo and radiation therapies as they are denied of passively diffused chemotherapeutic drugs due to poor vascularisation and high interstitial pressure. Furthermore, the deficiency in oxygen radicals also contributes to chemotherapy and radiotherapy resistance and decreasing DNA damage over the course of the treatment.
When a diagnosis is made during the early stage, a curative surgery is the first choice of therapy to remove the cancer followed by radiotherapy and/or chemotherapy to prevent reoccurrence. However, due to lack of specific symptoms in the clinical setting, up to 85% of patients with solid tumours present at an advanced stage where curative surgery is no longer an option. Chemo/radiotherapy or combinations of both were often used as palliative therapy, which fails to improve the overall survival rate of these patients. Chemicals/drugs in the form of antibodies and signal transduction inhibitors have been trialed in preclinical animals for late stage cancer therapy but fail to penetrate solid tumours, unable to accumulate into high concentration, inefficient in reaching a sufficient number of cancer cells, and leaving non-cancer stromal cells, stem cells, and tumour tissue structure untouched. Furthermore, frequent emergence of resistance renders these molecular therapies mostly ineffective.
Gene therapy to treat cancer is considered a better option than the above-mentioned drug therapies. Live virotherapy is considered to be the best approach currently in existence for treating advanced stage solid tumours but have disadvantages such as poor spread through tumors (Shayakhmetov et al., 2002) and rapid hepatic clearance from the circulation after intravenous delivery. Taken together, all of the current available anti-cancer therapies for reducing solid tumours have not met the need for an effective treatment of late stage cancer therapy until now.
Where the tumour central environments, also known as microenvironments, are detrimental for current chemo, radiation, and viro therapies, such environments can provide a unique opportunity for bacterial-based therapy that uses anaerobic bacteria and their innate oncolytic properties. Various anaerobic bacteria have been studied including strains belonging to the genus Clostridium. Naturally, Clostridium has two growth states; one is vegetative rod, and the other round spore. The latter is a tough, protective form of Clostridial bacterium which helps them survive through difficult times. A spore has a hard coating, wrapping around the outside of the key vital parts of the inside of the bacteria. In addition, there are also membrane layers lining the inside of the wrappings. Together the hard coating and the membrane lining enable the Clostridium to survive in tough environments that do not suit the vegetative rod. When growth conditions such as anaerobic environment and more nutrients become abundant, spore will increase gene expression and facilitate regrowth of vegetative rods.
Strict anaerobic proteolytic Clostridium sporogenes, C. novyi (include a novyi-NT with a major toxicin eliminated, but still has several other toxins and toxic factors) and C. sordellii have been shown to specifically target the unique hypoxic/necrotic tumour interior (see FIG. 8), causing modest oncolysis in several cancers when combined with vessel destroying agents. However, these strains are pathogenic microbes, causing various human diseases naturally in which their innate toxicity to humans has caused major issues. These issues are (1) toxicity levels are too high to be acceptable as appropriate therapeutic models for human treatment and (2) the biosafety implication for the use of a pathogenic bacterium for human is difficult to overcome. Furthermore, the wild type strains of Clostridium novyi and C. sordellii displayed rapid toxicity, resulting in significant death in experimental mice.
The present invention is predicated on the discovery and creation of derived, strictly anaerobic Clostridium ghonii strains that have been adapted to possess slower but assured oncolysis and, importantly and surprisingly, are relatively non toxic compared to other clostridial strains. The newly discovered strains are derivatives of an avirulent, non-pathogenic strain of Clostridium ghonii. These derived strains of Clostridium ghonii are not just useful alternatives to Clostridial strains in the state of the art (e.g. C. sporogenes, C. novyi and C sordeili), but display fewer toxic side effects than these other Clostridial strains. Without being bound by any biological mechanism or process, the novel adaptation process developed by the inventor and described herein enables the new derived bacterial strains to gain additional ability to leak through defect vessels in the angiogenic/hypoxic/necrotic regions of the solid tumour after intravenous injection of clostridial spores of the present invention, germinate into rod, efficiently and indiscriminately lyse/liquefy various cells as well as the tumour structure (fibronectins and collagens) inside the tumour, thus fundamentally destroying and altering tumour microenvironment. This destruction of the tumour microenvironment effectively arrests tumour growth possibly followed by tumour regression and in some instances eventually leading to the nearly complete or fully complete destruction of the entire tumour. The present invention creates opportunities to combine the approach of using injectable spores with existing approaches to ensure complete elimination of the tumour. As a non-limiting example, one such opportunity is the use of a targeted monoclonal antibody, which otherwise is not effective in the treatment of solid tumour. When a targeted monoclonal antibody is used in a tumour which has part or all of its microenvironment destroyed by the new derived bacterial strains of the present invention, it becomes far more potent because of the newly changed tumour microenvironment. Use of spores of the present invention as described and defined is herein is a clear contribution over the state of the art whether in the form of a single cancer therapy or in combination with another cancer therapy.